Radioimaging and radiochemotherapy phenolic thioether amines and acyl derivatives thereof for use in diagnosing and treating pigmentation disorders

ABSTRACT

Compounds useful in radioimaging and radiochemotherapy as it particularly relates to the treatment and location of malignant melanomas and other pigmenting disorders, as well as novel acyl derivatives of phenolic thioester amines and their use in compositions for blocking melanin synthesis in human or animal melanocyte cells.

FIELD OF THE INVENTION

This invention relates to compounds which are particularly useful inradioimaging and radiochemotherapy as it particularly relates to thetreatment and location of malignant melanomas and other pigmentingdisorders. Novel acyl derivatives of phenolic thioether amines and theiruse in compositions for blocking melanin synthesis in human or animalmelanocyte cells are also described. More particularly, the compositionsinvolving the novel derivatives are useful in treating pigmentationproblems due to a variety of skin disorders, including skin cancer inthe form of melanoma.

BACKGROUND OF THE INVENTION

Phenylthioalkylamines is a broad class of compounds having a variety ofuses which include therapeutics. U.S. Pat. Nos. 4,134,996 and 4,183,927disclose certain phenylthioalkylamine compounds which are useful asplatelet aggregation inhibitors. Other phenylthioalkylamine compounds ofapplicant's published International application WO91/16302 are useful asdepigmenting agents in treating a variety of pigmentary diseases. Suchdiseases are often characterized in the elevated levels of the enzymetyrosinase in melanocytes; i.e., human and animal cells which synthesizethe pigment melanin. There are a variety of pigmentary diseases, such asmelasma, melanoma, moles and the like. In particular, moles aresusceptible to becoming melanoma after exposure to sunlight whichprecipitates increased synthesis of tyrosinase.

Usually commercial forms of depigmenting compositions are based on theuse of hydroquinone. However, the hydroquinone preparations are veryunstable and cause skin irritation. Hydroquinone compositions can alsocause permanent whitening of the skin if used for a prolonged period andat a high concentration. As to treatment of melanoma, this is presentlyattended to by surgical procedures, since any type of known non-surgicaltreatment of melanoma is unsatisfactory.

Research work has been conducted in the field of phenolic and diphenoliccompounds to serve as a basis for chemotherapeutic treatment of melanomaand skin depigmentation. In particular, 4-S-cysteinylphenol (4-S-CP) and4-S-cysteaminylphenol (4-S-CAP) have been synthesized and evaluated forcytotoxicity to normal epidermal melanocytes to determine theireffectiveness as depigmenting agents and antimelanoma agents. Miura etal, "Synthesis of Cysteinylphenol, Cysteaminylphenol, and RelatedCompounds, and In Vivo Evaluation of Antimelanoma Effect", Arch.Dermatol Res. (1987) 279:219-225, disclose the effect of 4-S-CAP and4-S-CP in depigmentation of black hair follicles as manifested by lossof functioning melanocytes. It was established that 4-S-CAP was a potentagent in prolonging the lifespan of melanoma-bearing mice and henceexhibited inhibition of melanoma growth. 4-S-CP and the methyl ester of4-S-CP also exhibited some inhibition of melanoma growth, although notas active as 4-S-CAP.

The same compounds, 4-S-CP and 4-S-CAP, were also investigated forproperties of depigmentation of black guinea pig skin by topicalapplication. The results of this work is reported by Ito et al,"Depigmentation of Black Guinea Pig Skin by Topical Application ofCysteaminylphenol, Cysteinylphenol, and Related Compounds", The Journalof Investigative Dermatology, Vol. 88 No. 1, Jan. 1987. Although 4-S-CAPdemonstrated depigmenting properties, inflammatory changes of the skinof the guinea pigs was prominent. 4-S-CAP was capable, however, of:

1. decreasing the number of functioning melanocytes;

2. decreasing the amount of epidermal melanin pigments; and

3. degenerating and destroying melanocytes.

4-S-CP and 4-S-CAP were also investigated for their selectivecytotoxicity on follicular melanocytes. This was reported by Ito et al,"Selective Cytotoxicity of 4-S-Cysteaminylphenol on FollicularMelanocyte of the Black Mouse: Rational Basis for its Application toMelanoma Chemotherapy", Cancer Research, Jun. 15, 1987 47:3278-3284. Itwas reported that 4-S-CAP demonstrated cytotoxicity in thedepigmentation of black hair follicles, whereas it had no effect on thealbino follicles. Hence 4-S-CAP is actively engaged in the melaninsynthesis of the melanocytes.

4-S-CAP, however, has several limitations from the standpoint ofpractical clinical use. These limitations include:

a. hypotensive effect; and

b. high toxicity due to 4-S-CAP being a substrate for monoamineoxidase(MAO) which in the plasma converts 4-S-CAP into an aldehyde form whichproduces a non-specific cytotoxicity.

Homologs of 4-S-CAP have been investigated for antimelanoma anddepigmenting properties. As reported in Alena et al, Melanocytotoxicityand Antimelanoma Effects of Phenolic Amine Compounds in Mice In Vivo,Cancer Research 50:3743-3747, Jun. 15, 1990, such homologs demonstratedepigmentation properties and N-acetyl-4-S-CAP demonstrates potentantimelanoma properties. The benefits and uses of these homologs andrelated compounds for use as depigmenting agents and antimelanoma agentsare described in applicant's published International application WO91/16302. Although the compounds disclosed in that application areeffective depigmenting agents as well as antimelanoma agents, there arestability and toxicity problems associated with some of the compounds.In particular the very effective N-acetyl-4-S-CAP compound tends to beunstable and can cause topical irritations on administration.

As noted, melanin synthesis is a biological property unique to thepigment cell, melanocyte and its neoplastic counterpart, malignantmelanoma. In order to develop a targeted chemotherapy of malignantmelanoma, phenolic and catecholic melanin precursors have beensynthesized and their melanocytotoxicity and antimelanoma effects havebeen examined (Ito, Y. et al, 1987; Miura, S. et al, 1987; Alena, F. etal, 1990; Jimbow, K. et al, 1992). Among these, S-substituted phenolicamines, N-acetyl-4-S-cysteaminylphenol (N-Ac-4-S-CAP) was found to be asuperior substrate of melanin synthesizing enzyme, tyrosinase(Pankovich, J. et al, 1990; Miura, T. et al, 1990), and to possess themost effective melanocytotoxic activity and antimelanoma effect (Miura,T. et al, 1990; Alena, F. et al, 1990; Wong, M. et al, 1991). 4-S-CAPwas, however, as noted, above, found to have some general cytotoxicity(Alena, F. et al, 1990), because it is metabolically transformed acytotoxic aldehyde metabolite by plasm monoamine oxidase (Pankovich, J.et al; Inoue, S. et al, 1990). On the other hand, N-Ac-4-S-CAP has shownan absence of such cytotoxicity (Alena, F. et al, 1990), indicating thatN-Ac-4-S-CAP may be a compound suitable for the development of melanomachemotherapy.

In the treatment of various pigmentary disorders, it would also bebeneficial to determine the specificity of the compounds used in thetreatment and in particular, compounds such as N-Ac-4-S-CAP. In thisregard, we have also discovered that certain anti-melanoma anddepigmenting agents can be labelled with radioactive molecules toachieve not only radioimaging but as well, radio-chemotherapyparticularly in the treatment of malignant melanoma.

In accordance with this invention, we have also discovered esterifiedcompounds which exhibit excellent stability and very low generaltoxicity, but have selective toxicity for melanocytes.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, there is provided acompound for use in radioimaging and radiochemotherapy in the diagnosisand/or treatment of melanoma and other pigmentary disorders, saidcompound being selected from the group of compounds represented by theformula: ##STR1## wherein R₁ is H or C₁ -C₈ alkyl;

R₂ is H or C₁ -C₈ alkyl;

R₃ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl;

R₄ is H or C₁ -C₈ alkanoyl;

Ra is a radioactive material useful in radioimaging and/orradiochemotherapy where the dotted line (---) indicates:

1) Ra is covalently bound to the structure of formula (I);

2) Ra is ionically associated with the structure of formula (I); or

3) Ra is radioactive carbon which is part of the structure of formula(I);

and

x is 1 to 5,

with the provisos that when x is 1, one of R₁, R₂ or R₃ is other than H,and that the sulphur containing group and the Ra group are in the 2, 4or 6 positions of the phenyl ring.

In accordance with a further aspect of the invention, there is provideda process for radioimaging melanoma colonies in vivo in a subjectcomprising:

i) administering to the subject's circulatory system one or moreradioimaging compounds selected from the group of compounds representedby the formula: ##STR2## wherein R₁ is H or C₁ -C₈ alkyl;

R₂ is H pr C₁ -C₈ alkyl;

R₃ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl;

R₄ is H or C₁ -C₈ alkanoyl;

Ra is a radioactive material useful in radioimaging and/orradiochemotherapy where the dotted line (---) indicates:

1) Ra is covalently bound to the structure of formula (I);

2) Ra is ionically associated with the structure of formula (I); or

3) Ra is radioactive carbon which is part of the structure of formula(I);

and

x is 1 to 5,

with the provisos that when x is 1, one of R₁, R₂ or R₃ is other than H,and that the sulphur containing group and the Ra group are in the 2, 4or 6 positions of the phenyl ring; and

ii) detecting the presence of emitted radiation from an accumulation ofsaid selected radioimaging compound as said administered compound bindssolely to any melanoma tissue present in said subject.

In accordance with another aspect of the invention, there is provided aprocess for radiochemotherapy treatment of melanoma cells in a subjectcomprising:

i) administering to the subject's circulatory system one or more of theradiochemotherapy compounds selected from the group of compoundsrepresented by the formula: ##STR3## wherein R₁ is H or C₁ -C₈ alkyl;

R₂ is H pr C₁ -C₈ alkyl;

R₃ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl;

R₄ is H or C₁ -C₈ alkanoyl;

Ra is a radioactive material useful in radioimaging and/orradiochemotherapy where the dotted line (---) indicates:

1) Ra is covalently bound to the structure of formula (I);

2) Ra is ionically associated with the structure of formula (I); or

3) Ra is radioactive carbon which is part of the structure of formula(I);

and

x is 1 to 5,

with the provisos that when x is 1, one of R₁, R₂ or R₃ is other than H,and that the sulphur containing group and the Ra group are in the 2, 4or 6 positions of the phenyl ring.

In accordance with another aspect of the invention, the preferredcompounds of formula (I) are when X is 1 and R₄ is alkanoyl.

In accordance with another aspect of the invention, a compound of theformula (II): ##STR4## wherein R₁ is H or C₁ -C₈ alkyl;

R₂ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl;

R₃ is H or C₁ -C₈ alkyl;

R₄ is C₁ -C₈ alkanoyl; and

x is 1 to 5;

with the proviso that when x is 1 one of R₁, R₂ or R₃ is other than H,and that the sulphur containing group is in the 2, 4 or 6 position ofthe phenyl ring.

In accordance with another aspect of the invention, the preferredcompounds of formula (II) are when x is 1 and R₄ is alkanoyl.

In accordance with a further aspect of the invention, a pharmaceuticalcomposition comprises a compound of formula (II) and a pharmacologicallyor biologically acceptable carrier.

According to a preferred aspect of the invention, this composition isparticularly useful as a depigmenting composition in sunscreeninglotions. The composition is also useful as an antimelanoma agent anduseful for the treatment of melasma.

In accordance with another aspect of this invention, a compositionuseful for blocking melanin synthesis in human or animal melanocytecells, comprises:

i) a biologically effective amount of an active compound selected fromthe group represented by formula (II); and

ii) a suitable biologically compatible carrier for the selected activecompound.

According to another aspect of the invention, a method for blockingmelanin synthesis in human or animal melanocyte cells comprises treatinghuman or animal skin by use of a composition comprising a biologicallyeffective amount of an active compound selected from the grouprepresented by formula (II).

Such methods of treatment are preferably applied in the depigmenting ofskin due to UV exposure, melasma and is also useful in the treatment ofmelanoma.

According to another aspect of the invention, there is provided a methodof formulating a composition useful for blocking melanin synthesis inhuman or animal melanocyte cells of skin comprises mixing:

i) a biologically effective amount of an active compound selected fromthe group represented by formula (II); and

ii) a suitable biologically compatible carrier.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The radiolabelled compounds of this invention are capable of binding tomelanoma tissue such that when the tissue is subjected to radioimagingthe existence of such melanoma colonies or cells appears in the image.Further, by virtue of the binding of these compounds to melanoma sites,the radioactive material may be used in radiochemotherapy treatment ofthe melanoma cells to either kill such cells or effect their remission.

The preferred compounds of this invention exhibit considerably lesstoxicity than known forms of phenolic amine compounds such as theaforementioned 4-S-CP and 4-S-CAP. In particular, when 4-S-CAP is usedin radioimaging or radiochemotherapy, the compound collects in the eyeas well as in the melanoma tissue. However, the compounds of formula (I)do not have such drawbacks and are therefore particularly suited to thisnew use. When the compounds of the formula (I) are used inradiochemotherapy they may be combined with suitable pharmaceuticalexcipients and carriers wherein the radioactive selected compounds arepresent in a biologically effective amount in the carriers. Suitablebiologically, compatible carriers may be solutions when injected byeither the IP or SC route. Suitable biologically, compatible carriersfor injection include physiologically normal saline and other types ofreadily injectable solutions which are well understood by those skilledin the art and readily available. The preferred injectable carrier is aneutral buffer having a pH of 7 to 7.4 and does not in any way effectthe activity of the radiolabelled compounds. For purposes of injection,the concentration of the radiolabelled compound in the injectablesolution is normally in the range of 200 to 1200 mg per kg of bodyweight where the preferred dosage is in the range of 300 mg to 500 mgper kg of body weight. In the following examples, further information isprovided as to the amount of compounds injected for purposes ofradioimaging.

Although one skilled in the art appreciates that there are a variety oftechniques for making the compounds of formula (I) before they areradiolabelled the proposed method for manufacturing the compounds is viathe Weirmeister reaction as described in WO 91/16302.

As will be demonstrated in the following examples, radiolabelledcompounds of this invention bind specifically to melanoma tissues andthe radiolabelled compound or radiolabelled derivatives thereof remainin the melanoma tissue for a sufficient period of time for eithertherapeutic treatment or for subsequent radioimaging. The radiolabellingof the compound occurs normally in the phenyl ring in accordance withthe following reaction schemes. ##STR5## wherein I^(Ra) is a radioactiveform of iodine.

Exemplary iodine labelling is as follows:

N-acetyl-3-iodo-4-S-cysteaminyl-phenol;

N-acetyl-2-S-cysteaminyl-3-iodo-phenol;

N-acetyl-2-S-cysteaminyl-5-iodo-phenol;

N-{2-{(2'-iodo-4'-acetoxy-phenyl)thio}ethyl}-acetamide;

N-{2-{(2'-acetoxy-5'-iodo-phenyl)thio}ethyl}-acetamide; and

N-{2-{(2'-acetoxy-3'-iodo-phenyl)thio}ethyl}-acetamide.

Various radiolabelling agents may be used such as carbon 14, U-14-C,iodine (such as ¹²³ I, ¹²⁵ I and ¹³¹ I), technetium (such as ^(99m) Tc),fluorine (such as ¹⁸ F), indium (such as ¹¹¹ In) and 35S for example.The method of introduction of the radiolabelling agent to the compoundwill depend on the choice of radiolabelling agent, as one skilled in theart would appreciate. The radiolabelling agent will be associated withthe structure of the compound through one of the several ways: (1)through a covalent bond with the compound structure; (2) through ionicassociation with the compound structure; or (3) as part of the structureof the compound.

Novel acylated compounds of this invention are represented by thecompounds of formula (II). It has been found that those compounds areparticularly useful in the treatment of various pigmentation disordersand, when formulated into suitable pharmaceutical compositions, exhibitexcellent stability and very low general toxicity, but selectivetoxicity with respect to melanocytes. Hence, in accordance with anotheraspect of this invention, the pharmaceutical compositions are veryuseful in the treatment of pigmentary diseases such as treatmentsinvolving one or more of the following:

blocking melanin synthesis in human or animal melanocyte cells,

inhibiting the metabolic pathway involving tyrosinase in human or animalmelanocyte cells, and treatment of melanoma, treatment ofhyperpigmentation and treatment of cutaneous hyperpigmentation.

In formulae (I) and (II), preferred lower alkyl and lower alkanoylgroups are C₁ to C₆. The most preferred lower alkyl and lower alkanoylgroups are C₁ to C₄.

Preferred compounds of formula (I) includeN-acetyl-4-S-cysteaminylphenol, N,N-dimethyl-4-S-cysteaminylphenol,4-S-homo-cysteaminylphenol, α-methyl-4-S-cysteaminylphenol,N-{2-{(4'-acetoxyphenyl)-thio)ethyl)-acetamide, N-{1-methyl{2-{(4'-acetoxyphenyl)thio}ethyl}-acetamide, N,N-dimethyl(2-{(4'-acetoxyphenyl)-thio}ethylamine orN-propionyl-4-S-cysteaminylphenol.

Preferred specific compounds of the invention of Formula (II) are:

    ______________________________________    Compound        R.sub.1 R.sub.2                                   R.sub.3                                          R.sub.4    ______________________________________    1. N-(1-Methyl-{2-{(4'-                    CH.sub.3                            H      COCH.sub.3                                          COCH.sub.3    acetoxyphenyl)thio}-    ethyl}acetamide    2. N,N,-Dimethyl {2-{(4-                    H       CH.sub.3                                   CH.sub.3                                          COCH.sub.3    acetoxyphenyl)thio}-    ethylamine    3. N-{2-{(4'-acetoxy-                    H       H      COCH.sub.3                                          COCH.sub.3    phenyl)thio}ethyl}-    acetamide    4. N-{2-{(4-acetoxyphenyl)-                    H       H      COC.sub.2 H.sub.3                                          COCH.sub.3    thio}ethyl}propionamide    ______________________________________

The compounds of formula (II) may be manufactured by a variety ofsynthesis techniques. Generally, they may be prepared by acylation ofthe corresponding cysteaminylphenol compounds.

The cysteaminylphenol compounds may also be prepared in accordance withthe technique disclosed by Miura et al, "Synthesis of Cysteinylphenol,Cysteaminylphenol, and Related Compounds, and in vivo Evaluation ofAntimelanoma Effect", Arch. Dermatol. Res. (1987) 279:219-225. Thisentails the reaction of a phenol with a cysteamine to yield4-S-cysteaminylphenol or the 2-S-cysteaminylphenol.4-S-cysteaminylphenol may then be isolated from the reaction product bySilica gel column chromatography or crystallization with changing pH ofsolution. It is understood that the various derivatives of thisinvention may be made by various radical substitutions to the sulphurchain, or substituting a thiol with the desired chain.

Details of processes for making some of the preferred compounds of theinvention are set out in the accompanying Examples.

The intermediate cysteaminylphenol compounds may be acylated by reactionwith the appropriate alkanoyl; for example, acetyl chloride may bereacted with N-{2-((4-hydroxyphenyl)thio)ethyl}acetamide, to give thepreferred compound of the inventionN-{2-((4'-acetoxyphenyl)-thio)ethyl}acetamide (NAP-TEA).

The compositions of this invention having active agents of formula (II)provide a mechanism for treating a variety of pigmentary diseases, suchas melasma and other hyperpigmentation diseases and melanoma and otherskin cancers and for preventing melasma and skin cancers which arenormally induced by exposure to UV radiation. This activity of thecomposition is achieved by the selected active compound of thecomposition becoming toxic in the melanocytes in the presence oftyrosinase without being a substrate for plasma MAO. The compositionsupon administration, according to this invention, have significantmelanoma cytotoxicity and antimelanoma effects. This is recognized inthe significant depigmentation of hair on black experimental animals, aswell as significantly inhibiting the experimental growth of lungmetastases of B16 F10 melanoma cells. The compositions of this inventionhave also demonstrated selective toxicity to other neural crest tumors,such as pheochromocytoma and neuroblastoma cells.

Experimental tests on animals indicate per single dose toxicity levelsin the range of 1,400 mg/kg of body weight for the preferred compound offormula (II), NAP-TEA.

In the pharmaceutical compositions of the invention, the term "abiologically effective amount of the active compound" means that asufficient amount of the compound in the composition is provided suchthat upon administration to the human or animal by, for example, i.p.,s.c. or topical route provides sufficient active agent on eachapplication to give the desired result in treating various pigmentarydiseases, including melanoma, melasma, or acting as UV blocker and/ordepigmenting agent in sunscreen lotions. However, the biologicallyeffective amount of the active compound is at a level that it is nottoxic to the human or animal during the term of treatment.

The term "a suitable biologically compatible carrier" includes a solventfor the compound when the compound is injected by either i.p. or s.c.route. Suitable biologically compatible carriers for injection includephysiologically normal saline and other types of readily injectablesolutions which are well understood by those skilled in the art. Thepreferred injectable carrier is a neutral buffer having a pH of 7.0 to7.4. When the compound is topically applied, the carrier may be any typeof suitable excipient or carrier in the form of cosmetic compositions,pharmaceutical adjuvants, sunscreen lotions and the like.

Suitable carriers for topical application include conventional skintreatment compositions, such as cosmetic compositions and pharmaceuticalpreparations. Examples of such ingredients for topical application areoils such as liquid paraffin, vaseline, methyl-polysiloxane, castor oil,squalane, and coconut oil; anti-oxidants such as butylatedhydroxyanisole, butylhydroxytoluene, ethyl gallate, and tocopherol;surfactants such as sodium laureate, laurylpyridinium chloride,polyoxyethylene sorbitan monooleate, glyceryl monoarachate, sodiumN-stearyl-N, N-dimethyl glycine, oleyl hydrolysed animal protein, andpolyoxypropylene glyceryl ether phosphate; humectants such as glycerol,sodium 2-pyrrolidone-5-carboxylate, and sodium lactate; thickeners suchan tragacanth gum, quince seed gum, zanthan gum, carboxyvinyl polymerand bentonite; preservatives such as benzoic acid, allylp-hydroxybenzoates, dehydroacetic acid and trichlorocarbonilide;coloring agents and pigments such as Acid Red Rhodamine B, Violamin R,Orange SS, Naphthol, Yellow-S, Tartrazine, Alizarin, Cyanine Green F,Brilliant Blue CFC, Acid Violet, Carthamine, β-carotene, Red, Blue &Yellow Oxide of Iron, titanium dioxide, Yellow Iron Oxide, Cobalt Blue,Ultramarine Blue, Rose & Violet, tri-iron tetroxide, and carbon black;waxes such as Bees wax, Japan wax, Carnauba wax, Candelilla wax, andlanolin; film-forming agents such as nitrocellulose and polyvinylalcohol; solvents or dispersing media such a water and alcohols (e.g.ethanol); powders such as aluminum powder, talc, kaolin, zinc oxide,titanium dioxide, mica, calcium, carbonate, and treated powders;plasticizers such as acetyl tributyl citrate, and dibutylphthalate;pharmaceutically active agents such as retinol, palmitate; γ-orizanol,pyridoxine dipalmitate, ascrobyl dipalmitate, ergocalciferol,di-α-tocopherol acetate, biotin, ethinylestradial estrone,hydrocrontisone, calcium pantothenate, ammonium glycyrrhizinate,allantoid, quaiasulene, and hinokitiol; and perfumes such as musk,civet, amber, jasmine absolute, and rose oil.

The skin treatment compositions, according to this invention, can beprepared in any conventional form, for example, solubilized forms suchas cosmetic lotions, and emulsified forms such as liquid creams, creams,ointments and dispersions.

The pharmaceutical compositions of this invention are particularlyuseful for depigmenting skin where the composition is applied topicallyor injected by s.c. or i.p. route to the afflicted area to achievedesired degree of depigmentation of the skin. Hence as another aspect ofthe invention, the composition is useful in the method of treating skinfor purposes of depigmentation. As well, the invention provides forformulating a composition which is useful for depigmenting skin.

The compositions of the invention are also especially useful fortreating melanoma. The composition is administered either topically orby injection through i.p. or s.c. route. The composition is used intreating melanoma to either clear it up or place it in remission.Furthermore, a method of the invention is in the formulating of thecomposition useful for treating melanoma.

The compounds of formula (II) may be used in sunscreening lotions asdepigmenting agents. It has been found that the active agents of formula(II):

1. prevent UV radiation activating melanin synthesis in melanocytes;

2. prevent moles from becoming melanoma after exposure to sunlight; and

3. are substrates for the enzyme tyrosinase where it is thought thatenzymatic conversion of the substrates produce components which act asdepigmenting agents and possibly as UV blockers in the sunscreen lotion.

Quite surprisingly, we have found that, upon administration of theacylated derivatives of formula (II), the compounds are deacylated tobecome potent melanocytotoxic and depigmenting agents. Such in vivoconversion of the compounds is extremely beneficial from the standpointof product stability and reduced general toxicity. Once the compounds offormula (II) are deacylated upon administration, they become excellenttyrosinase substrates. According to a preferred embodiment of theinvention, NAP-TEA, once incorporated into the body, is deacylated tobecome N-acetyl-4-S-CAP which is, as already demonstrated in theaforementioned International Application WO91/16302, a strong tyrosinasesubstrate that is present only in pigment cells. This enzyme activitybecomes highly expressed in malignant melanoma and melasma cells wherethe specificity of N-acetyl-4-S-CAP is important in avoiding generaltoxicity to the patient. The therapeutic approach in the use of thesecompounds for pigmentary diseases such as melasma and skin cancer, suchas malignant melanoma, have become very important as well as preventivemedicine for photoaging and skin cancer developed after exposure tosunlight. Melanoma represents 2% of all cancer by instance and isgenerally increasing in the population at a rate exceeded only by lungcancer.

For purposes of treatment, it is understood that depending upon theroute of administration the composition of this invention may havevarious concentrations of active agent in the biologically compatiblecarrier. For purposes of injection, the concentration of the activeagent in the injectable solution usually is in the range of 200 to 1200mg/kg of body weight with a preferred dosage in the range of 300 mg to500 mg/kg of body weight. For purposes of topical application, theconcentration of the active agent in the cosmetic cream or the likeusually is in the range of 4% to 10% by weight in a cream base with apreferred composition in the range of 4% to 6% by weight in a creambase.

It is appreciated that various complementary agents may be included withthe active agents of this invention. For example, the composition may beadministered in combination with L-dopa and/or antidecarboxylase. Suchcombinations may have selected use in the treatment of variousmelanomas.

The following Examples demonstrate various aspects of the invention inproviding the diagnosis and treatment of pigmentation diseases,depigmenting properties and antimelanoma properties. The variouscompounds of formula (I) may be radiolabelled in accordance with apreferred procedure outlined in the following Examples. The procedureshave been described in particular reference to the compoundN-acetyl-4-S-CAP; however, it is understood that the procedure may besimilarly applied to the other compounds of formula (I). Furthermore,the Examples also demonstrate effectiveness in blocking melaninsynthesis for only one of the compounds of formula (II). However, it isunderstood that the compounds of formula (II) all exhibit in apredictable way similar properties.

EXAMPLE 1 Synthesis of N-Acetyl-4-S-cysteaminyl (U-¹⁴ C)phenol

(U-¹⁴ C)phenol (9.26 MBq, 333.3 MBq/mmol) in toluene (0.25 mL) was addedto aqueous HBr (47% w/v; 0.5 mL solution containing unlabelled phenol(1.7 mg; 0.018 mmol) and cystamine•2HCl (45 mg; 0.2 mmol). The mixturewas heated for 30 min at 130° C. in a screw-capped reaction vial. Aftercooling, the solvent was removed under vacuum and the residue wasdissolved in aqueous methanol (3% v/v). The solution was applied to aSep-Pak C₁₈ cartridge. The cartridge was washed with aqueous methanol(10% v/v; 0.6 mL for removal of excess cystamine and (¹⁴ C) labelled4-S-CAP and 2-S-CAP were eluted with aqueous methanol (20% v/v; 3.0 mL).The eluent was concentrated under vacuum. The major compounds wereseparated on preparative TLC using the mobile phase I. The products wereextracted from TLC with MeOH in CHCl₃ (20% v/v). The two isomers wereacetylated with acetic anhydride in dry pyridine at 25° C. for 24 hours.The solvent was removed under vacuum. The residue was dissolved in NH₃(2.0 M) and stirred at 25° C. for 30 min for O-deacetylation. Afterreaction, the methanol solvent was removed under vacuum and the residuewas purified on preparative TLC using mobile phase II. (¹⁴C)N-Ac-4-S-CAP (9.5 μCi, 351.9 kBq) and (¹⁴ C)N-Ac-2-S-CAP(5.7 μCi,211.1 kBq) in 3.8% and 2.3% radiochemical yield respectively. Theradiochemical purity of the two isomers was more than 99%.

This Example demonstrates that one is able to synthesize (¹⁴C)N-Ac-4-S-CAP with conventional acetyacetylation and O-deacylationafter electrophilic substitution on phenol of the NH₂ CH₂ CH₂ S⁺ cationin HBr solution, as per the following reaction scheme. ##STR6##

In the above reaction process, (¹⁴ C)-acetyl-2-S-cysteaminylphenol ((¹⁴C)-Ac-2-S-CAP was also obtained as a minor isomer. The synthesis of (¹⁴C)4-S-CAP using the above reaction was as described previously (Somayajiet al, 1989). It was difficult to remove cystamine by methanolprecipitation or to separate the 2 and 4 isomers of CAP by TLC system.However, it was possible to remove cystamine and accomplish preliminarypurification using a Sep-pak C¹⁸ cartridge. The chromatographicseparation of the two isomers was accomplished with mobile phase I. Thisbasic mobile phase allowed a complete separation of the two isomeric CAPcompounds of the reaction mixture (4-S-CAP:Rf=0.66; 2-S-CAP:Rf=0.53).

Unlabelled crystalline phenol (2 mg) reacted smoothly with cystamine inHBr solution in a small scale reaction. An overall yield wasapproximately 76% as a mixture of the two isomers, the ratio of 4-S-CAPto 2-S-CAP being 2.5. Acetylation of the O-position and the N-positionon isomers was carried out with acetic anhydride in dry pyridine.O-Deacetylation for the synthesis of N-Ac-4-S-CAP was performed with NH₃in methanol (2.0 M) under mild conditions without N-deacetylation. Amixture of 4-S-CAP (a major product) and 2-S-CAP (a minor product)containing the ¹⁴ C-radiolabel were obtained by using a two phasereaction system (toluene:HBr) under reflux, and with the addition ofunlabelled phenol this gave low yield of products. If a source of (U¹⁴C) phenol were available as a solid material or as a more concentratedsolution, the yield and the specific activity can be greatly increased.(¹⁴ C)N-Ac-4-S-CAP synthesized from (¹⁴ C)4-S-CAP gave a highradiochemical yield (68%). Although the specific activity of (¹⁴C)N-Ac-4-S-CAP and (¹⁴ C)N-Ac-2-S-CAP was low (450 kBq/μmol), theproducts would be sufficient to carry out experimental studies. The useof solid (U¹⁴ C)phenol with a high specific activity for the synthesisof (¹⁴ C)4-S-CAP and (¹⁴ C)2-S-CAP would increase both the specificactivity and the yield of the final products.

All chemicals used were of reagent grade quality. (U-¹⁴ C)Phenol intoluene was purchased from Sigma Chemical Company (Saint Louis, Mo.,U.S.A.). Preliminary purification was performed on Sep-Pak C₁₈Cartridges (Waters Chromatography Division, Millipore Corporation,Milford, U.S.A.). Whatman PLK5F preparative silica gel 150A plates wereused for separation of 4-S-cysteaminyl (U-¹⁴ C)phenol, (¹⁴ C) 4-S-CAPand 2-cysteaminyl (U-¹⁴ C) phenol ((¹⁴ C)2-S-CAP) using mobile phase I(CHCl₃ MeOH:NH₄ OH=24:12:1. 1 v/v/v) and using mobile phase II (CHCl₃:MeOH=85 15 v/v) for the purification of the N-acetyl derivatives of thetwo isomers. ¹⁴ C-Radioactivity determination of products was performedin Aquasal®-2 (Biotechnology Systems NEN® Research Product, Boston,Mass., U.S.A.) with a Beckman LS 3801 liquid scintillation spectrometer.HPLC analyses were carried out with a Waters system consisting of aModel 600E system controller, Model U6K injector and Model 441 detectorat 254 nm, using μBonapak C₁₈ reverse phase compression (10μ) column(Waters Millipore Canada Ltd., Mississauga, Ontario) and MeOH:0.1%acetic acid in H₂ O (1:1 v/v) as eluent with a flow rate of 1 mL/min.The final products, N-acetyl-4-S-cysteaminyl (U-¹⁴ C) phenol ((¹⁴ C)N-Ac-4-S-CAP) and N-acetyl-2-S-cysteaminyl (U-¹⁴ C)phenol ((¹⁴ C)N-Ac-2-S-CAP) were confirmed by comparing their retention times to thoseof authentic unlabelled compounds on HPLC. Radiochemical purity afterTLC was determined by collect eluent fractions (0.5 mL) from an HPLCanalysis and counting by a liquid scintillation spectrometer.

EXAMPLE 2A Synthesis of N-{2-((4-Acetoxyphenyl)-thio)ethyl}acetamide(NAP-TEA)

Procedure

Acetyl chloride method

To solution of N-{2-((4-Hydroxyphenyl)thio)ethyl} acetamide (200 mg,0.95 mmol) in dry pyridine was added slowly acetyl chloride at -15° C.with stirring. The resulting solution was stirred at 25° C. for 1.5hour. The residue obtained after removal of solvent was chromatographedon a low pressure silica gel column (CANAG, D-O, fine grain; Mobilephase: 5% MeOH/CHCL₃). The fractions containing product were collectedand then the white powder obtained after removal of solvent wasrecrystallized to gain the white crystals from H₂ O-MeOH. (Chemicalyield: 217 mg, 0.85 mmol 90.3% ) mp=87-88°

Characterization

¹ H NMR (Acetone-d₆); δ 7.52 (bro, s, 1H, NH), 7.41 (d, fine structure,2H, J=8.5 Hz, aromatic C(2)-H, C(6)-H), 7.04 (d, fine structure 2H,J=8.5 Hz, aromatic C(3)-H, C(5)-H), 3.33 (m, 2H, J(CH₂ --CH₂)=6 Hz,J(CH₂ --NH)=7.5 Hz, CH₂ CH₂ NH), 3.05 (dd, 2H, J(CH_(2-CH) 2)=8 Hz,SCH₂), 2.22(s, 3H, CH₃ COO), 1.83 (s, 3H, CH₃ CONH).

¹³ C NMR (Acetone-d₆) ppm; 169.95 (1C, CH₃ CONH) 169.29 (1C, CH₃ COO),150.12 (1C, aromatic, C(4)), 133.61 (1C, aromatic, C(1)), 130.76 (2C,aromatic, CH) 122.98 (2C, aromatic, CH), 39.35 (1C, CH₂ NH), 33.48 (1C,SCH₂), 22.52 (1C, CH₃ CONH), 20.61 (1C, CH₃ COO).

Exact mass calc. for C₁₁ H₁₅ NO₃ S:253.07725; found (HRMS), 253.0773;intensity=18.18%.

EXAMPLE 2B Synthesis of N-{2-{(4-Acetoxyphenyl)thio}-ethyl}propionamide(NAP-TEP)

A mixture of 2-ethyl-2-oxazoline (786 mg, 7.94 mmol) and4-hydroxythiophenol (1 g, 7.94 mmol) was heated at 125° for 2 h under Argas. After cooling of the reaction mixture, the mixture was evaporatedunder vacuum. The residue was purified with a low pressure normal phasesilica gel column chromatography (mobile phase: 10% MeOF in CHCl₃).N-propionyl-4-S-cysteaminylphenol (N-Pro-4-S-CAP) was obtained as awhite powder on evaporation of the solvent (Chemical yield: 53.4%, 953mg).

A mixture of N-propionyl-4-S-cysteaminylphenol, dry pyridine, and aceticanhydride is then stirred at 25° for 24 hours. The reaction mixture isevaporated under vacuum and the residue is purified with a low pressurenormal phase silica gel column chromatography (mobile phase: 8% MeOH inCHCl₃). N-{2-{(4-acetoxyphenyl)thio}-ethyl}-propionamide (NAP-TEP) isobtained as a white powder upon evaporation of the solvent.

EXAMPLE 3

In vivo covalent binding of (¹⁴ C)-N-acetyl-4-S-CAP was investigated inmice. Female C57BL/6J mice received a single intraperitoneal injectionof 2.0 mmol/kg (¹⁴ C)-N-acetyl-4-S-CAP compound. The animals were killed48 hours later. The tissues were removed and the covalent binding wasdetermined as follows.

Five different tissues were examined for covalent binding ofradioactively labelled N-acetyl-4-S-CAP intermediate(s). The maximumbinding of N-acetyl-4-S-CAP was detected in the s.c. melanoma tumor andmelanoma colony-bearing lung tissues. In contrast, the normal organs,e.g., the lung without melanoma colonies, kidney and liver, which areactively involved in drug metabolism of N-acetyl-4-S-CAP, did not reveala significant accumulation of radioactive material. The results of suchstudy are set out in the following Table 1.

                  TABLE 1    ______________________________________    In Vivo Covalent Binding of (14C)-NACAP to Lung with and    without B16F10 Melanoma Colonies, B16F10 Melanoma s.c.    Tumor Tissue, Liver and Kidney                  Convalent Binding.sup.b    Tissue.sup.a  (nmol covalently bound/kg of protein    ______________________________________    Lungs with B16F10                  0.330 + 0.113    melanoma colonies    Lungs without B16F10                  0.010 + 0.001    melanoma colonies    B16F10 melanoma                  0.521 + 0.076    s.c. tumor    Liver         0.013 + 0.003    Kidney        0.008 + 0.001    ______________________________________     .sup.a Female C57BL/6J mice received a single intraperitoneal injection o     2.0 nmol/kg (.sup.14 C)NACAP. The animals were killed 48 hr later, the     tissues were removed, and the covalent binding was determined as describe     under above.     .sup.b The data are presented as the means + S.D. (n = 3).

In vivo biodistribution by whole-body autoradiography was determined inuse of the compounds of this invention and in particular, (¹⁴C)N-acetyl-4-S-CAP at 48 hours after i.p. injection of (¹⁴C)N-acetyl-4-S-CAP in the mouse, radioactive material was cleared fromthe body and was not detectable in any normal organs except the lumen ofthe large intestine. A whole-body autoradiogram of the mouse with bothan s.c. B16F10 melanoma tumor and a B16F10 lung melanoma colonies aftersingle i.p. injection of (¹⁴ C)N-acetyl-4-S-CAP (5.0 μCi) showedradioactivity in infected organs. 48 hours after injection the animalwas killed, frozen and prepared for sectioning. The 20-μm sections werefreeze-dried and exposed to x-ray film for whole-body autoradiography.

The result demonstrates the significant detoxication of N-acetyl-4-S-CAPin the liver followed by the excretion of N-acetyl-4-S-CAP metabolitesinto the bile. The s.c. melanoma tumor and the lung with melanomacolonies were the only organs displaying a significant accumulation ofthe radioactivity.

The radiolabelled compounds of this invention and in particular,radiolabelled N-acetyl-4-S-CAP and radiolabelled NAP-TEA areparticularly useful as radioimaging agents without causing toxic sideeffects in the host. Also, in appropriate dosages the compounds may beused effect radio-chemotherapy at specific melanoma sites within thehost.

EXAMPLE 4 - Experiments for O-Deacylation of NAP-TEA in Mouse Plasma

NAP-TEA was introduced to mouse plasma to determine by in vitroexperimentation the extent to which NAP-TEA is deacetylated to formN-Acetyl-4-S-CAP. The procedure was as follows. Into 100 μl of mouseplasma, 10 μg of NAP-TEA was introduced. The mixture was shook forone-half of a minute and then passed through a Sep-Pak C₁₈ cartridge ineither 7 ml of 5% methanol/water to form eluent 1 or in 4 ml of 50%methanol/water to form eluent 2. Both eluents 1 and eluents 2 werecollected. Aqueous methanol was removed from eluent 2 to provide aresidue which was subjected to HPLC under the following conditions. TheHPLC had a column of μ Bonapak C₁₈ under reverse phase radicalcompression. The residue was mixed with methanol:water at 50:50 with aflow rate through the radiograph at 1 ml/min. The detector was a WaterAssociates Model 441 with detection by ultraviolet at 250 nm. All thatwas detected in eluent 2 as passed through the chromatograph wasN-Acetyl-4-S-CAP which indicates that NAP-TEA was readily deacylated bythe mouse plasma to give the desired active agent.

EXAMPLE 5 - Determination of NAP-TEA, N-Acetyl-4-S-CAP and Metabolitesin In Vivo Experimentation

A C57BL/6J Black Mouse was injected at 300 mg/kg body weight by i.p.administration of NAP-TEA. 100 μl of plasma was extracted from the mouseafter 5 minutes of injection and after 20 minutes of injection. Theplasma samples were treated in accordance with the procedure of Example2. The plasma samples were passed through a Sep-Pak C₁₈ cartridge ineither 5% methanol:water to produce eluent 1 or 50% methanol/water toproduce eluent 2. Eluent 2 had the aqueous methanol removed therefrom toprovide a residue. The residue was redissolved in methanol/0.1% aceticacid and water in a ratio of 50:50 and subjected to HPLC for both the 5minute and 20 minute plasma samples. Their was no evidence of NAP-TEAbut their was evidence of N-Acetyl-4-S-CAP. The concentration ofN-Acetyl-4-S-CAP in the 5 minute extracted sample being considerablygreater than in the 20 minute extracted sample to indicate that in vivoNAP-TEA is deacylated to N-Acetyl-4-S-CAP which in turn is then taken upby the mouse melanocytes.

EXAMPLE 6 - In Vitro Cytotoxicity of NAP-TEA

MTT assay in accordance with the standard procedure

MTT solution (Sigma catalog No. M2128) was dissolved in PBS at 1 mg/mland filtered to sterilize the solution. The solution was stored in thedark at 4° C. until used. The growing cells were harvested, counted andinoculated into 96 well microtiter plates (1-2×10³ cells/well). After 24h, drugs were applied to culture wells, and the cultures were incubatedfor 5 days at 37° C. At this time, 50 μl of MTT solution was added tomicroculture wells. After 4 h incubation at 37° C., supernatant wasremoved from each well. 150 μl of DMSO was added to solubilized theMTT-formazan product. After mixing gently, absorbance at 540 nm wasmeasured with ELISA plate spectrophotometer.

Results

NAP-TEA showed lower in vitro cytotoxicity than that of N-Acetyl-4-S-CAPon 3 cell lines.

    ______________________________________                   Compound                   IC.sub.50    Cell line        NAP-TEA  N-Acetyl-4-S-CAP    ______________________________________    HeLa             77 μg/ml                              75 μg/ml    SK-MEL-23        31 μg/ml                              25 μg/ml    UT amelanotic melanoma                     14 μg/ml                              12 μg/ml    ______________________________________

EXAMPLE 7 - Utilization of NAP-TEA with Mushroom Tyrosinase

Procedure

A reaction mixture consisting of NAP-TEA (500 μmol) and of mushroomtyrosinase (11 units) in 1.0 ml of 0.05 M sodium phosphate buffer (pH6.8) was incubated at 37° C. in a water bath. The reaction was stoppedperiodically by cooling with ice. Aliquots removed at set time intervalswere rapidly cooled to 0° C. in an ice bath and the remaining NAP-TEAwas measured using HPLC.

HPLC instrument and conditions

Water 600 E liquid chromatograph system

Conditions:

μBondapak C₁₈, Radial-Pak Cartridge (Waters)

Mobile phase; 70% MeOH/H₂ O, 1 ml/min

Detector; Waters Model 441 UV absorbance

detector

Detection; Wavelength 254 nm

Results

NAP-TEA was not a substrate of mushroom tyrosinase, due to the blockingeffect of acetoxy group on the 4-position of the benzene ring becauseits concentration remained constant in the reaction mixture. However,the concentration of N-Acetyl-4-S-CAP in the reaction mixture droppedoff rapidly over 25 minutes to almost zero amount after 50 minutes inthe reaction mixture.

EXAMPLE 8 - Melanocytotoxicity In Vivo of NAP-TEA (Depigmentation ofBlack Hair)

Hairs were plucked manually from the back of 6-8 week old C57BL/6J blackmice (4 mice). Starting on day 2, daily for 14 days NAP-TEA was injectedi.p. The dose was 300 mg/kg body weight.

Results

After completion of the i.p. injections of NAP-TEA, mice weredepigmented with the new replacement hair being almost pure white.

EXAMPLE 9 - Depigmenting Effect of NAP-TEA on Newborn Black Mice

Six newborn (3 day old) C57BL/6J black mice were injected i.p. with asingle dose of 0.1 mL of NAP-TEA solution (300 mg/kg body weight).

Results

The newborn mice were depigmented in all hair follicles with the growinghair being light grey after a single i.p. injection of NAP-TEA.

EXAMPLE 10 - In Vivo Melanocytotoxicity of NAP-TEA in Pig

Solutions of 15% NAP-TEA in DMSO(w/v) and of 15% NAP-TEA in 60%EtOH(w/v) were prepared for topical application to a Yucatan pig.NAP-TEA was applied to the back skin of the pig twice a day for 8 weeks.After 8 weeks, punch biopsy was performed from the application area andfrom normal skin. Specimens obtained were kept in 2N NaBr solution for 3hours followed by separation of the epidermis from dermis. Specimens ofepidermis were incubated for 3 hours in phosphate buffer (pH 7.4)containing L-Dopa. The specimens were fixed in 10% neutral formalinsolution and then washed twice in PBS. Finally, the specimens weremounted in glycerol gel. The numbers of Dopa-positive melanocytes in 9adjacent 1 mm² fields from each sample were counted.

Results

The fields from skin samples treated with NAP-TEA revealed a significantreduction in the number of functioning melanocytes;

    ______________________________________                  Number of functioning melanocytes                  (Average of 9 fields)    ______________________________________    Normal epidermis                    166 ± 74/mm.sup.2    NAP-TEA in DMSO  32 ± 14/mm.sup.2    NAP-TEA in 60% EtOH                     74 ± 25/mm.sup.2    DMSO only       114 ± 56/mm.sup.2    EtOH only       117 ± 38/mm.sup.2    ______________________________________

From the above specific examples, it is apparent that one of thepreferred compounds, according to this invention, is converted in vivoto N-Acetyl-4-S-CAP which functions, as already discussed in applicant'sco-pending International Application WO91/16302 as an excellenttherapeutic agent in the treatment of pigmentary diseases. It istherefore understood that the acylated forms of the compounds of thisinvention are altered in vivo to yield compounds which already have beenestablished as excellent therapeutic agents in the field of treatingpigmentary disorders.

Throughout the specification we have referenced several Journal articleswhich relate to standard procedures and techniques. Those articles arelisted as follows.

1. Alena F., Jimbow K. and Ito S. (1990) Melanocytotoxicity andAntimelanoma effects of Phenolic Amine Compounds in Mice in vivo. CancerRes. 50, 3743.

2. Ito Y. and Jimbow K. (1987) Selective Cytotoxicity of4-S-cysteaminylphenol on Follicular Melanocytes of the BlackMouse--Rational Basis for its Application to Melanoma Chemotherapy.Cancer Res. 47, 3278.

3. Inoue S., Ito S., Wakamatsu K., Jimbow K. and Fujita K. (1990)Mechanism of Growth Inhibition of Melanoma Cells by4-S-cysteaminylphenol and its Analogues. Biochem. Pharmacol. 39, 1077.

4. Jimbow K., Iwashina T., Alena F., Yamada K., Pankovich J. and UmemuraT. (1992) Exploitation of Pigment Biosynthesis Pathway as a SelectiveChemotherapeutic Approach for Malignant Melanoma. J. Invest. Dermatol.(in press).

5. Miura S., Ueda T., Jimbow K., Ito S. and Fujita K. (1987) Synthesisof Cysteinylphenol, Cysteaminylphenol and Related Compounds, and in vivoEvaluation of Antimelanoma Effect. Arch. Dermatol. Res. 279, 219.

6. Miura T., Jimbow K. and Ito S. (1990) The in vivo Antimelanoma Effectof 4-S-cysteaminylphenol and its N-acetyl derivative. Int. J. Cancer 46,931.

7. Pankovich, J. Jimbow K. and Ito S. (1990) 4-S-cysteaminylphenol andits Analogues as Substrates for Tyrosinase and Monoamine Oxidase.Pigment Cells Res. 3, 146.

8. Somayaji V.V., Wiebe, L.I. and Jimbow K. (1989) ExperimentalAntimelanoma Agents: The Synthesis of 4-S-cysteaminylphenol (U-¹⁴C)phenol. Nuc. Compact 20, 158.

9. Wong M. and Jimbow K. (1991) Selective Cytotoxicity ofN-acetyl-4-S-cysteaminylphenol on Follicular Melanocytes of Black Mice.Br. J. Dermatol 124, 56.

Although preferred embodiments of the invention are described herein indetail, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claim.

I claim:
 1. A compound selected from the group of compounds representedby the formula: ##STR7## wherein R₁ is H or C₁ -C₈ alkyl;R₂ is H; R₃ isH, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl; R₄ is C₁ -C₈ alkanoyl;and whereinthe compound of Formula (I) is radiolabelled with a radioactive elementor a compound comprising a radioactive element and wherein saidradioactive element or compound comprising a radioactive element 1) iscovalently bound to the structure of formula (I); 2) is ionicallyassociated with the structure of formula (I); or 3) is radioactivecarbon which is part of the structure of formula (I); and x is 1 to 5,with the provisos that when x is 1, at least one of R₁ and R₃ is otherthan H, and that the sulphur containing group and the radioactiveelement or compound comprising a radioactive element are in the 2, 4 or6 positions of the phenyl ring.
 2. A process for radioimaging melanomacolonies in vivo in a subject comprising:i) administering to thesubject's circulatory system one or more radioimaging compounds selectedfrom the group of compounds represented by the formula: ##STR8## whereinR₁ is H or C₁ -C₈ alkyl; R₂ is H; R₃ is H, C₁ -C₈ alkyl or C₁ -C₈alkanoyl; R₄ is C₁ -C₈ alkanoyl;and wherein the compound of Formula (I)is radiolabelled with a radioactive element or a compound comprising aradioactive element and wherein said radioactive element or compoundcomprising a radioactive element 1) is covalently bound to the structureof formula (I); 2) is ionically associated with the structure of formula(I); or 3) is radioactive carbon which is part of the structure offormula (I);and x is 1 to 5, with the provisos that when x is 1, atleast one of R₁, and R₃ is other than H, and that the sulphur containinggroup and the radioactive element or compound comprising a radioactiveelement are in the 2, 4 or 6 positions of the phenyl ring; and ii)detecting the presence of emitted radiation from an accumulation of saidselected radioimaging compound as said administered compounds bindssolely to any melanoma tissue present in said subject.
 3. A process forradiochemotherapy treatment of melanoma cells in a subject comprising:i)administering to the subject's circulatory system one or moreradiochemotherapy compounds selected from the group of compoundsrepresented by the formula; ##STR9## wherein R₁ is H or C₁ -C₈ alkyl; R₂is H; R₃ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl; R₄ is C₁ -C₈alkanoyl;and wherein the compound of Formula (I) is radiolabelled with aradioactive element or a compound comprising a radioactive element andwherein said radioactive element or compound comprising a radioactiveelement 1) is covalently bound to the structure of formula(I); 2) isionically associated with the structure of formula (I); or 3) isradioactive carbon which is part of the structure of formula (I);and xis 1 to 5, with the provisos that when x is 1, at least one of R₁, andR₃ is other than H, and that the sulphur containing group and theradioactive element or compound comprising a radioactive element are inthe 2, 4 or 6 positions of the phenyl ring.
 4. A compound of claim 1wherein said sulphur containing group is in the 4 position.
 5. Acompound of claim 4 wherein said radioactive element or compoundcomprising a radioactive element is in the 2 or 6 position of the phenylgroup.
 6. A compound of claim 1, selected from the group consisting ofradiolabelled N-{2-{(4'-acetoxyphenyl)thio}ethyl}-acetamide andradiolabelled N-{1-methyl {2-{(4-acetoxyphenyl)thio}ethyl}-acetamide. 7.A compound of claim 6 wherein said selected compound is radiolabelledN-{2-{(4-acetoxyphenyl)thio}ethyl}-acetamide.
 8. A compound as claimedin claim 1 wherein said radioactive element is selected from the groupconsisting of carbon 14, U-14-C, iodine, technetium, fluorine, indiumand ³⁵ S.
 9. A compound of the formula (II): ##STR10## wherein R₁ is Hor C₁ -C₈ alkyl;R₂ is H; R₃ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl; R₄ isC₁ -C₈ alkanoyl; and x is 1 to 5; andwith the proviso that when x is 1at least one of R₁ and R₃ is other than H, and that the sulphurcontaining group is in the 2, 4 or 6 position of the phenyl ring.
 10. Apharmaceutical composition comprising a compound of claim 9 and apharmaceutically acceptable carrier.
 11. A compound of claim 9 whereinsaid sulphur group is in the 4 position.
 12. A compound of claim 9,wherein said compound is N-{2-{(4'-acetoxyphenyl)thio}ethyl}-acetamide.13. A compound of claim 9, wherein said compound isN-(1-methyl{2-{(4'-acetoxyphenyl)thio}ethyl)-acetamide.
 14. A compoundof claim 9, wherein said compound isN-{2-{(4-acetoxyphenyl)thio}ethyl}-propionamide.
 15. A pharmaceuticalcomposition as claimed in claim 10 wherein said carrier is a topicalvehicle.
 16. A pharmaceutical composition as claimed in claim 10 whereinsaid carrier is a sunscreening composition.
 17. A process of claimwherein said sulphur containing group is in the 4 position.
 18. Aprocess of claim 3 wherein said sulphur containing group is in the 4position.
 19. A process of claim 17 wherein said radioactive element orcompound comprising a radioactive element is in the 2 or 6 position ofthe phenyl group.
 20. A process of claim 18 wherein said radioactiveelement or compound comprising a radioactive element is in the 2 or 6position of the phenyl group.
 21. A process of claim 2 wherein saidcompounds are selected from compounds derived fromN-{2-{(4'-acetoxyphenyl)thio}ethyl}-acetamide or N-{1-methyl{2-{(4-acetoxyphenyl)thio}ethyl}-acetamide.
 22. A process of claim 3wherein said compounds are selected from compounds derived fromN-{2-{(4'-acetoxyphenyl)thio}ethyl}-acetamide or N-{1-methyl{2-{(4-acetoxyphenyl)thio}ethyl}-acetamide.
 23. A composition of claim10 wherein said sulphur group is in the 4 position.
 24. A composition ofclaim 10 comprising N-{2-{(4'-acetoxyphenyl)thio}ethyl}-acetamide.
 25. Acomposition of claim 10 comprisingN-(1-methyl{2-(4'-acetoxyphenyl)thio}ethyl}-acetamide.
 26. A compositionof claim 10 comprising N-{2-{(4-acetoxyphenyl)thio}ethyl}propionamide.27. A process of claim 21 wherein said compound is radiolabelledN-{2-{(4-acetoxyphenyl)thio}ethyl}-acetamide.
 28. A process of claim 22wherein said compound is radiolabelledN-{2-{(4-acetoxyphenyl)thio}ethyl}-acetamide.
 29. A method of blockingmelanin synthesis in human or animal melanocyte cells comprisingadministering to a patient a melanin synthesis blocking amount of acomposition comprising a compound of formula (II): ##STR11## wherein R₁is H or C_(1-C) ₈ alkyl;R₂ is H or C_(1-C) ₈ alkyl; R₃ is H, C_(1-C) ₈alkyl or C₁ -C₈ alkanoyl; R₄ is C_(1-C) ₈ alkanoyl; and x is 1 to 5;andwith the proviso that when x is 1 at least one of R₁, R₂ and R₃ isother than H, and that the sulphur containing group is in the 2, 4 or 6position of the phenyl ring.
 30. A method of inhibiting the metabolicpathway involving tyrosinase in human and animal melanocyte cellscomprising administering to a patient an inhibiting amount of acomposition comprising a compound of the formula (II): ##STR12## whereinR₁ is H or C₁ -C₈ alkyl;R₂ is H or C₁ -C₈ alkyl; R₃ is H, C₁ -C₈ alkylor C₁ -C₈ alkanoyl; R₄ is C₁ -C₈ alkanoyl; and x is 1 to 5; andwith theproviso that when x is 1 at least one of R₁, R₂ and R₃ is other than H,and that the sulphur containing group is in the 2, 4 or 6 position ofthe phenyl ring.
 31. A method of therapeutically treating melanomacomprising administering to a patient a therapeutically effective amountof a composition comprising a compound of the formula (II): ##STR13##wherein R₁ is H or C₁ -C₈ alkyl;R₂ is H or C₁ -C₈ alkyl; R₃ is H, C₁ -C₈alkyl or C₁ -C₈ alkanoyl; R₄ is C₁ -C₈ alkanoyl; and x is 1 to 5;andwith the proviso that when x is 1 one of R₁, R₂ or R₃ is other thanH, and that the sulphur containing group is in the 2, 4 or 6 position ofthe phenyl ring.
 32. A method of therapeutically treatinghyperpigmentation comprising administering to a patient atherapeutically effective amount of a composition comprising a compoundof the formula (II): ##STR14## wherein R₁ is H or C₁ -C₈ alkyl;R₂ is Hor C₁ -C₈ alkyl; R₃ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl; R₄ is C₁ -C₈alkanoyl; and x is 1 to 5; andwith the proviso that when x is at least 1of R₁, R₂ and R₃ is other than H, and that the sulphur containing groupis in the 2, 4 or 6 position of the phenyl ring.
 33. A method oftherapeutically treating cutaneous hyperpigmentation comprisingadministering to a patient a therapeutically effective amount of acomposition of the formula (II): ##STR15## wherein R₁ is H or C₁ -C₈alkyl;R₂ is H or C₁ -C₈ alkyl; R₃ is H, C₁ -C₈ alkyl or C₁ -C₈ alkanoyl;R₄ is C₁ -C₈ alkanoyl; and x is 1 to 5; andwith the proviso that when xis at least 1 one of R₁, R₂ and R₃ is other than H, and that the sulphurcontaining group is in the 2, 4 or 6 position of the phenyl ring.
 34. Amethod of claim 29 wherein R₂ is H.
 35. A method of claim 30 wherein R₂is H.
 36. A method of claim 31 wherein R₂ is H.
 37. A method of claim 32wherein R₂ is H.
 38. A method of claim 33 wherein R₂ is H.
 39. Acompound as claimed in claim 8 wherein said radioactive element isselected from the group consisting of ¹²³ I, ¹²⁵ I, ¹³¹ I, ^(99m) Tc, ¹⁸F and ¹¹¹ In.